UHRA does not alter purified fibrin clot morphology or fiber diameter. Clots were made by incubating 3 mg/mL human fibrinogen in 3.0 mM CaCl2 plus UHRA or protamine and then initiating clotting with 2.5 National Institutes of Health Units/mL thrombin. Clots were then allowed to mature for 1 hour and processed for SEM imaging. (A) SEM images of fibrin clots formed in the presence of UHRA at different concentrations (50-500 µg/mL) were determined at both low (original magnification ×10 000; scale bar, 2 µm) and high (original magnification ×25 000; scale bar, 1 µm) magnifications. Clot architectures formed in the presence of UHRA are similar to that for the polycation-free control, even up to UHRA concentrations of 500 µg/mL. (B) SEM images of fibrin clots formed in the presence of protamine exhibit altered morphologies compared with the control clot. (C) Fibrin fiber diameters of clots formed in the presence of UHRA or protamine. Fiber diameter is measured from scanning electron micrographs, using ImageJ software. A total of 30 fibers were analyzed. Fibers for size analysis were selected by probing 4 different spots in each image. Data are mean ± SE (n = 30 fibers; measured from 4 images of 2 independent clots). Statistical significance for fiber diameter was determined by comparing the UHRA or protamine-treated group with the control, using a 1-way ANOVA followed by a Dunnett post hoc test. Fibrin fibers formed in the presence of 25 µg/mL protamine are significantly thicker than those in the control clot (***P < .001).
